Edward Smith

The aromatic amino acid tryptophan is the main precursor for indole-3-acetic acid (IAA), which involves various parallel routes in plants, with indole-3-acetaldoxime (IAOx) being one of the most common intermediates. Auxin signaling is well known to interact with free radical nitric oxide (NO) to perform a more complex effect, including the regulation of root organogenesis and nitrogen nutrition. To fathom the link between IAA and NO, we use a metabolomic approach to analyze the contents of… Show more

The aromatic amino acid tryptophan is the main precursor for indole-3-acetic acid (IAA), which involves various parallel routes in plants, with indole-3-acetaldoxime (IAOx) being one of the most common intermediates. Auxin signaling is well known to interact with free radical nitric oxide (NO) to perform a more complex effect, including the regulation of root organogenesis and nitrogen nutrition. To fathom the link between IAA and NO, we use a metabolomic approach to analyze the contents of low-molecular-mass molecules in cultured cells of Arabidopsis thaliana after the application of S-nitrosoglutathione (GSNO), an NO donor or IAOx. We separated the crude extracts of the plant cells through ion-exchange columns, and subsequent fractions were analyzed by gas chromatography-mass spectrometry (GC-MS), thus identifying 26 compounds. A principal component analysis (PCA) was performed on N-metabolism-related compounds, as classified by the Kyoto Encyclopedia of Genes and Genomes (KEGG). The differences observed between controls and treatments are mainly explained by the differences in Trp contents, which are much higher in controls. Thus, the Trp is a shared response in both auxin- and NO-mediated signaling, evidencing some common signaling mechanism to both GSNO and IAOx. The differences in the low-molecular-mass-identified compounds between GSNO- and IAOx-treated cells are mainly explained by their concentrations in benzenepropanoic acid, which is highly associated with IAA levels, and salicylic acid, which is related to glutathione. These results show that the contents in Trp can be a marker for the study of auxin and NO signaling. Show less

The pyridine nucleotides nicotinamide adenine dinucleotide [NAD(H)] and nicotinamide adenine dinucleotide phosphate [NADP(H)] simultaneously act as energy transducers, signalling molecules, and redox couples. Recent research into photosynthetic optimisation, photorespiration, immunity, hypoxia/oxygen signalling, development, and post-harvest metabolism have all identified pyridine nucleotides as key metabolites. Further understanding will require accurate description of NAD(P)(H) metabolism… Show more

The pyridine nucleotides nicotinamide adenine dinucleotide [NAD(H)] and nicotinamide adenine dinucleotide phosphate [NADP(H)] simultaneously act as energy transducers, signalling molecules, and redox couples. Recent research into photosynthetic optimisation, photorespiration, immunity, hypoxia/oxygen signalling, development, and post-harvest metabolism have all identified pyridine nucleotides as key metabolites. Further understanding will require accurate description of NAD(P)(H) metabolism, and genetically encoded fluorescent biosensors have recently become available for this purpose. Although these biosensors have begun to provide novel biological insights, their limitations must be considered and the information they provide appropriately interpreted. We provide a framework for understanding NAD(P)(H) metabolism and explore what fluorescent biosensors can, and cannot, tell us about plant biology, looking ahead to the pressing questions that could be answered with further development of these tools. Show less

Reactive oxygen species (ROS) are produced throughout plant cells as a by-product of electron transfer processes. While highly oxidative and potentially damaging to a range of biomolecules, there exists a suite of ROS-scavenging antioxidant strategies that maintain a redox equilibrium. This balance can be disrupted in the event of cellular stress leading to increased ROS levels, which can act as a useful stress signal but, in excess, can result in cell damage and death. As crop plants become… Show more

Reactive oxygen species (ROS) are produced throughout plant cells as a by-product of electron transfer processes. While highly oxidative and potentially damaging to a range of biomolecules, there exists a suite of ROS-scavenging antioxidant strategies that maintain a redox equilibrium. This balance can be disrupted in the event of cellular stress leading to increased ROS levels, which can act as a useful stress signal but, in excess, can result in cell damage and death. As crop plants become exposed to greater degrees of multiple stresses due to climate change, efforts are ongoing to engineer plants with greater stress tolerance. It is therefore important to understand the pathways underpinning ROS-mediated signalling and damage, both through measuring ROS themselves and other indicators of redox imbalance. The highly reactive and transient nature of ROS makes this challenging to achieve, particularly in a way that is specific to individual ROS species. In this review, we describe the range of chemical and biological tools and techniques currently available for ROS and redox marker measurement in plant cells and tissues. We discuss the limitations inherent in current methodology and opportunities for advancement. Show less

Methylated flavones, commonly found in many plants of the Brassicaceae family, have potent antioxidant and anticancer activity with diverse therapeutic potential. However, the specific regioisomers of methylated flavones can have significantly different biochemical and potentially therapeutic properties as shown by various bioassays but analytically differentiating these compounds has been technically challenging and rarely reported. In this study, we demonstrate differentiation and… Show more

Methylated flavones, commonly found in many plants of the Brassicaceae family, have potent antioxidant and anticancer activity with diverse therapeutic potential. However, the specific regioisomers of methylated flavones can have significantly different biochemical and potentially therapeutic properties as shown by various bioassays but analytically differentiating these compounds has been technically challenging and rarely reported. In this study, we demonstrate differentiation and identification of selected bioactive methylated flavone regioisomers, namely 5,7,3′-trihydroxy-4′-methoxyflavone, and 5,7,4′-trihydroxy-3′-methoxyflavone extracted from Coronopus didymus, a member of the Brassicaceae family, using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC-ESI-QTOF-MS/MS). Characteristic MS/MS product ions produced from neutral loss of carbon monoxide, and a methyl radical from the [M-H]– ion, exhibited differential relative abundances attributed to different structural stabilities under the same activation and collision-induced dissociation conditions. MS/MS also provided structural information which was sufficient to differentiate the methylated regioisomers and determine the position of the methyl group based on interpretation of their respective fragmentation patterns. Quantification showed 5,7,4′-trihydroxy-3′-methoxyflavone was at least 1.60 mg per 10 g plant material in C. didymus extracts. This study demonstrates a straightforward and novel approach to rapidly differentiate, identify and quantify regio-isomeric methylated flavone natural products using reversed-phase UPLC-MS/MS. Show less

NADH and NAD are a ubiquitous cellular redox couple. Although the central role of NAD in plant metabolism and its regulatory role have been investigated extensively at the biochemical level, analyzing the subcellular redox dynamics of NAD in living plant tissues has been challenging. Here, we established live monitoring of NADH/NAD in plants using the genetically encoded fluorescent biosensor Peredox-mCherry. We established Peredox-mCherry lines of Arabidopsis (Arabidopsis thaliana) and… Show more

NADH and NAD are a ubiquitous cellular redox couple. Although the central role of NAD in plant metabolism and its regulatory role have been investigated extensively at the biochemical level, analyzing the subcellular redox dynamics of NAD in living plant tissues has been challenging. Here, we established live monitoring of NADH/NAD in plants using the genetically encoded fluorescent biosensor Peredox-mCherry. We established Peredox-mCherry lines of Arabidopsis (Arabidopsis thaliana) and validated the biophysical and biochemical properties of the sensor that are critical for in planta measurements, including specificity, pH stability, and reversibility. We generated an NAD redox atlas of the cytosol of living Arabidopsis seedlings that revealed pronounced differences in NAD redox status between different organs and tissues. Manipulating the metabolic status through dark-to-light transitions, respiratory inhibition, sugar supplementation, and elicitor exposure revealed a remarkable degree of plasticity of the cytosolic NAD redox status and demonstrated metabolic redox coupling between cell compartments in leaves. Finally, we used protein engineering to generate a sensor variant that expands the resolvable NAD redox range. In summary, we established a technique for in planta NAD redox monitoring to deliver important insight into the in vivo dynamics of plant cytosolic redox metabolism. Show less

NADPH is the primary source of cellular reductant for biosynthesis, and strategies for increasing productivity via metabolic engineering need to take account of the requirement for reducing power. In plants, while the oxidative pentose phosphate pathway is the most direct route for NADPH production in heterotrophic tissues, there is increasing evidence that other pathways make significant contributions to redox balance. Deuterium-based isotopic labelling strategies have recently been developed… Show more

NADPH is the primary source of cellular reductant for biosynthesis, and strategies for increasing productivity via metabolic engineering need to take account of the requirement for reducing power. In plants, while the oxidative pentose phosphate pathway is the most direct route for NADPH production in heterotrophic tissues, there is increasing evidence that other pathways make significant contributions to redox balance. Deuterium-based isotopic labelling strategies have recently been developed to quantify the relative production of NADPH from different pathways in mammalian cells, but the application of these methods to plants has not been critically evaluated. In this study, LC-MS was used to measure deuterium incorporation into metabolites extracted from heterotrophic Arabidopsis cell cultures grown on [1-2H]glucose or D2O. The results show that a high rate of flavin-enzyme-catalysed water exchange obscures labelling of NADPH from deuterated substrates and that this exchange cannot be accurately accounted for due to exchange between triose- and hexose-phosphates. In addition, the duplication of NADPH generating reactions between subcellular compartments can confound analysis based on whole cell extracts. Understanding how the structure of the metabolic network affects the applicability of deuterium labelling methods is a prerequisite for development of more effective flux determination strategies, ensuring data are both quantitative and representative of endogenous biological processes. Show less